Trisomy 21 results from partial or complete duplication of chromosome 21. This disorder results in intellectual disabilities, cardiac defects, and distinct craniofacial abnormalities. Several studies have investigated the genetic origin of these defects. A candidate gene is the dual specificity kinase, Dyrk1a. Dyrk1a is an attractive target, given that Dyrk1a haploinsufficiency results in craniofacial defects as well. While the craniofacial defects induced by these genetic imbalances are distinct, a common feature is midface hypoplasia. Previous work in our laboratory has established Xenopus laevis as ideal organism to study development of the midface and primary palate. Previous investigations, by our laboratory and others, suggest that dyrk1a is expressed in the Xenopus face. I hypothesize that Dyrk1a is an important regulator of midface development and a dosage imbalance of dyrk1a results in the distinct features present in Trisomy 21 and Dryk1a haploinsufficiency. To test this hypothesis, I will utilize Xenopus to examine the expression of dyrk1a during orofacial development. I will investigate the effects of decreased dyrk1a, via pharmacological inhibition with INDY, a Dyrk1a-specific inhibitor, or antisense oligonucleotide morpholinos (Dyrk1a MO). To assess the effects of overexpression I will inject a verified, full-length Xenopus dyrk1a mRNA construct. I will perform simple face measurements and geometric morphometrics to qualitatively and quantitatively investigate the effects of dyrk1a dosage imbalance. The cellular requirements and mechanisms of Dyrk1a signaling in the face remain to be elucidated. I will explore this by careful manipulation of the INDY treatment window and assessment of neural crest specification, migration, delamination, and differentiation. I expect to identify a role of Dyrk1a in one or more of these processes. To further investigate the requirements of Dyrk1a in the face, I will perform face transplantation assays with the dyrk1a MO. In Aim 2, I will investigate the role of retinoic acid (RA) in the regulation of Dyrk1a. Dyrk1a was identified our lab as a target of RA, in a large genomic screen of developing embryos with diminished RA signaling. A dosage imbalance of RA, whether increased or decreased, results in midfacial hypoplasia and a midfacial cleft. I hypothesize that RA inhibits the expression of dyrk1a. RA-mediated repression is known regulate levels of fgf8 in the developing embryo and preliminary data from our lab suggest that inhibition of RA can synergize with dyrk1a to produce facial abnormalities. I will examine dryk1a levels in an excess RA paradigm, and examine if artificially modulating RA levels induces the expected change in dyrk1a. If this is the case, increased RA will result in decreased dyrk1a and vice versa. I will conclude this aim by investigating the possibility of forcing the dosage imbalance, increasing the dosage of RA, and rescuing the midfacial phenotype induced by increased dyrk1a. These studies will further the understanding of the role of Dyrk1a during midfacial development and provide insight into the midfacial abnormalities associated with Trisomy 21 and Dyrk1a haploinsufficiency.